The distribution of neurons expressing NADPH diaphorase activity was examined histochemically in whole mount preparations of the neonatal guinea pig urinary bladder. NADPH diaphorase positive neurons were abundant in the intramural ganglia in both the detrusor and trigone regions of the bladder. Labelled nerve fibres were found in the ganglion interconnectives and in smooth muscle bundles. Mucosal epithelial cells and endothelial cells lining the blood vessels supplying the bladder were also found to express NADPH diaphorase activity. In order to verify that NADPH diaphorase activity represented the presence of nitric oxide synthase in bladder neurons, a well characterised tissue culture preparation was employed. This also provided an opportunity to estimate the proportion of the total population of bladder neurons which expressed NADPH diaphorase activity. Using a combination of histochemical and immunohistochemical techniques, NADPH diaphorase positive neurons were found to constitute approximately 90% of the total neuronal population, which was identified by labelling with an antiserum to the general neuronal marker protein gene product 9.5. Almost all neurons (99%) which expressed NADPH diaphorase activity in culture were also found to be immunoreactive for nitric oxide synthase. These findings indicate that nitric oxide may play a role in the neural control of bladder function, and this possibility is discussed.